Methods of decreasing or preventing pain using spicamycin derivatives

ABSTRACT

Methods of providing pain relief by administering a water-soluble derivative of spicamycin. Methods of using pain mediation agents are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application60/234,382, filed Sep. 20, 2000.

BACKGROUND OF THE INVENTION

Spicamycin (SPM) is an antitumor antibiotic produced by the bacteriumStreptomyces

alanosinicus is 879-MT₃ (Hayakawa et al., Agric. Biol. Chem.49:2685–2691, 1985). Spicamycin and its derivatives are also used forpain relief (U.S. Pat. No. 5,905,069). Naturally occurring SPM has thefollowing general structure, varying solely in the fatty acid moiety:

Formula I: Spicamycin

Synthetic variants of spicamycin and their use as an antitumor agent aredescribed in Otake et al., U.S. Pat. Nos. 5,461,036 and 5,631,238.

SUMMARY OF THE INVENTION

The invention is based on the unexpected discovery that substantialremoval of the fatty acid moiety in a subclass of spicamycin derivativesresults in drugs especially useful for the treatment of pain. Thissubclass of spicamycin derivatives exhibit increased water-solubilityrelative to fatty acid-containing derivatives, and are therefore moreamenable to formulations suitable for human administration (e.g.,formulations containing an aqueous physiological buffer). In addition,the potential toxicity of pharmaceutical formulations containing thissubclass of spicamycin derivatives can be reduced because no toxiclipophilic carriers are needed in order to solubilize the activeingredients.

Thus, the invention features a method of providing pain relief byidentifying a subject (e.g., a mammal, such as a human, dog, cat, orhorse) in need of pain relief, and administering to the subject anamount of a compound of Formula II effective to provide significant painrelief in the subject.

R₁ and R₂ are different from each other and represent —H or —OH, and Rrepresents (1) a substituted or unsubstituted alkyl having one or twocarbon atoms (i.e., no more than two carbon atoms), or (2) —H. Forexample, R can contain an amino group (e.g., a primary amino group), acarbonyl group or both. When R is —COCH₂NH₂, R₁ is —H, and R₂ is —OH,the compound is known as 4′-N-glycyl spicamycin amino nucleoside(SAN-Gly). When R is —H, the compound is known as SAN. See, e.g.,Kamishohara et al., Oncology Res. 6:383–390, 1994. A salt of thecompound of Formula II can also be used in the methods of the invention.

The compounds (and salts thereof) of Formula II useful in the methodsherein also are those where R₁ and R₂ are independently H or OH, whereinR₁ and R₂ are not simultaneously the same and:

Each R group can independently be an H or alkyl group having 1 to 5(e.g., 1 to 2, 1 to 3, 1 to 4) carbon atoms substituted with 1 to 3independent R³ or R⁴;

Each R³ is independently heterocyclyl or heteroaryl, either optionallysubstituted with 1–3 independent R⁵;

Each R⁴ is independently halogen, oxygen, sulfur, CF₃, SR⁶, OR⁶,OC(O)R⁶, NR⁶R⁶, NR⁶R⁷, COOR⁶, C(O)R⁶, or C(O)NR⁶R⁶;

Each R⁵is independently C1–C10 alkyl; halo; haloalkyl; SR⁶; OR⁶; NR⁶R⁶;COOR⁶; NO₂; CN; C(O)R⁶; C(O)NR⁶R⁶; OC(O)R⁶; S(O)₂R⁶; S(O)₂NR⁶R⁶;NR⁶C(O)N R⁶R⁶; NR⁶C(O)R⁶; NR⁶(COOR⁶); NR⁶C(O)R⁸; NR⁶S(O)₂NR⁶R⁶;NR⁶S(O)₂R⁶; NR⁶S(O)₂R⁸; or C1–C10 alkyl substituted with R⁴ or R⁸;

Each R⁶ is independently H, C1–C10 alkyl; C2–C10 alkenyl; C2–C10alkynyl; C3–C10 cycloalkyl; R⁸; or C1–C10 alkyl substituted with R⁸;

Each R⁷ is independently COOR⁹, C(O) NR⁹R⁹, S(O)₂R⁹; or S(O)₂NR⁹R⁹;

Each R⁸ is independently aryl, heteroaryl, or heterocyclyl;

Each R⁹ is independently H, C1–C10 alkyl, aryl, heteroaryl, orheterocyclyl.

The term “alkyl” denotes a straight or branched hydrocarbon chaincontaining carbon atoms or cyclic hydrocarbon moieties. These alkylgroups may also contain one or more double bonds or triple bonds. By“substituted alkyl” is meant an alkyl in which an atom of the alkyl issubstituted with, for example, a carbon, nitrogen, sulfur, oxygen, orhalogen atom, or alternatively a nitrogen, sulfur, oxygen, or halogenatom.

Examples of substituents that can be attached to any atom of the alkylgroup in a “substituted alkyl” include heterocyclyl groups; heteroarylgroups, amino groups, amido groups, alkoxy groups, acyloxy groups,thioalkoxy groups, acyl thioalkoxy groups, halogen groups, sulfonategroups, sulfonamide groups, ester groups, carboxylic acids, oxygen(e.g., a carbonyl group) and sulfur (e.g. a thiocarbonyl group).Substituents also include any chemical functional group that impartsimproved water-solubility to the molecule (e.g., carboxylic acid,carboxylic ester, carboxamido, morpholino, piperazinyl, imidazolyl,thiomorpholino, or tetrazolyl groups; both unsubstituted andsubstituted).

The terms “halo” and “halogen” refer to any radical of fluorine,chlorine, bromine or iodine. The terms “ring” and “ring system” refer toa ring comprising the delineated number of atoms, said atoms beingcarbon or, where indicated, a heteroatom such as nitrogen, oxygen orsulfur. The ring itself, as well as any substituents thereon, may beattached at any atom that allows a stable compound to be formed.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2 or 3 atoms of each ring may besubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like.

The term “heteroaryl” refers to an aromatic 5–8 membered monocyclic,8–12 membered bicyclic, or 11–14 membered tricyclic ring systemcomprising 1–3 heteroatoms if monocyclic, 1–6 heteroatoms if bicyclic,or 1–9 heteroatoms if tricyclic, said heteroatoms selected from O, N, orS, wherein 0, 1, 2 or 3 atoms of each ring may be substituted by asubstituent. Examples of heteroaryl groups include pyridyl, furyl orfuranyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl,quinolinyl, indolyl, thiazolyl, and the like.

The term “heterocyclyl” refers to a nonaromatic 5–8 membered monocyclic,8–12 membered bicyclic, or 11–14 membered tricyclic ring systemcomprising 1–3 heteroatoms if monocyclic, 1–6 heteroatoms if bicyclic,or 1–9 heteroatoms if tricyclic, said heteroatoms selected from O, N, orS, wherein 0, 1, 2 or 3 atoms of each ring may be substituted by asubstituent. Examples of heterocyclyl groups include piperizinyl,pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable”, as used herein, refers to compounds which possessstability sufficient to allow manufacture and which maintains theintegrity of the compound for a sufficient period of time to be usefulfor the purposes detailed herein (e.g., therapeutic or prophylacticadministration to a subject or antiseptic, wound dressing impregnation,sterilizant, or disinfectant applications).

A “subject in need of pain relief” does not necessarily experience paincurrently, and “pain relief” includes less than 100% reduction in pain.For example, the invention can be used to treat a mammal, including ahuman patient, a dog, a cat, or a horse, for neuropathic painattributable to any cause, e.g., postherpetic neuralgia, phantom oramputation stump pain, diabetic neuropathy, acquired immune deficiencysyndrome neuropathy, back pain, and visceral pain (e.g., chronicpancreatitis). By “neuropathic pain” is meant pain arising from injuryto or disturbance of the peripheral nervous system.

The compound can be administered locally or systemically, e.g., via animplant (for slow release, for example) or by intravenous bolusinjection or infusion. An “implant” is any device residing in a tissuedeeper than the skin, in which the device produces a regulated orcontinuous release of a compound. Such devices are well known in the artof drug delivery (see, e.g., U.S. Pat. No. 6,013,853). For example, acompound of Formula II can be prepared with carriers that will protectthe compound against rapid elimination from the body, such as acontrolled release formulation, including microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811. The amount of compound administered at one timecan be about 1 ng to 4 mg/m² body surface area (e.g., 80 ng to 1 mg/m²body surface area), and the compound can be formulated in an aqueoussolution that optionally contains pharmaceutically acceptable carriers.Other suitable dosages include about 1 to 1000 mg/kg body weight (e.g.,about 10 to 500, or about 100 mg/kg body weight).

Treatment in accordance with the invention produces relief of pain inpatients whose current pain is resistant to other methods of painrelief, such as using opioid drugs. The invention can also be used inanticipation of pain to prevent pain.

The invention further relates to methods of selectively inhibiting,treating, or preventing neuropathic pain selectively over acutenociceptive pain comprising identifying a subject in need of painrelief, and administering a compound (or composition) of any of theformulae delineated herein, including those of Formula II. In anotheraspect, the method involves selectively inhibiting, treating, orpreventing neuropathic pain selectively over nociceptive pain comprisingidentifying a subject in need of pain relief, and administering acompound (or composition) of any of the formulae delineated herein. Inone aspect of these methods, selectively refers to inhibitingneuropathic pain to a greater extent than nociceptive pain. In anotheraspect of these methods, selectively refers to inhibiting neuropathicpain at least 50% more (e.g., >100% more, >200% more, 500% more) thannociceptive pain as determined by standard pain models, including thosedelineated in Borsook et al., U.S. Pat. No. 5,905,069 (and referencescited therein), issued May 18, 1999, and Abdi et al., Anesth. Analg. 91,955–999 (2000).

The invention also relates to methods of assessing, identifying, orvalidating genes (or polypeptides encoded by those genes) as receptorsinvolved in pain mediation, including neuropathic pain treatment. Themethods comprise contacting a compound of any of the formulae hereinwith a test polypeptide and measuring the binding affinity of thecompound and test polypeptide. Those polypeptides (and the genesencoding them) having a greater affinity for the compound are candidateshaving a greater likelihood of being directly involved in painmediation, particularly neuropathic pain mediation. As such, they wouldbe interesting targets for research and development studies for newmechanisms of pain mediation and for targets of inhibition by ligands(e.g., peptide or small molecule drugs) for treatment or prevention ofpain. The analysis of the binding affinity of the compounds andreceptors can be performed using assays, methods, and techniques knownin the drug screening/design, genomics, and medicinal chemistry arts,including labeling (radiolabel, fluorescence) studies for detection ofcompound, target, or other ligand mediating a known peptide-ligandinteraction, or can involve an indirect read-out (e.g., measuringpresence of a marker whose release or formation is dependent upon thebinding interaction of the compound and test polypeptide).

The invention further relates to methods of assessing the efficacy ofpotential pain drugs (e.g., peptides, chemical entities, smallmolecules). Because the compounds of the formulae herein are effectivein mediating neuropathic pain, they are also useful as “standards” bywhich potential new pain drugs can be assessed. Such methods compriseevaluating a test compound (e.g., potential pain drug) against acompound of any the formulae herein (i.e., a standard), including acompound of Formula II, by subjecting the test compound and the compoundof any of the formulae herein to a subject or medium (e.g., patient,animal model, cell culture, in vitro assay) that provides measure orassessment of the effectiveness of the test compound and the compound ofany of the formulae herein in mediating pain or modulating the mechanismof pain. The method can further comprise evaluating the results of thecompound testing to assess the effectiveness of the test compound as apain drug. The measuring or assessing of the effectiveness of thecompounds in these methods can be performed by any number of appropriatetechniques and protocols known in the art and readily available.

The invention also relates to methods of identifying new genes,receptors, or peptides that are involved in mediation of pain. Thesemethods involve use of the compounds of any of the formulae herein,including those of Formula II, to investigate metabolic effects inducedby the compounds, including effects involved in pain mediation. Suchmethods comprise administering the compounds of any of the formulaeherein to a subject or medium (e.g., patient, animal model, cellculture, in vitro assay, tissue), collecting tissue from the subject ormedium (e.g., dorsal root ganglion, nerve tissue, spinal cord tissue, orcentral nervous system (CNS) tissue), and evaluating the tissue foreffects (e.g., identifying and/or quantifying: induction, suppression,indirect responses, marker production) of known or novel genes,receptors, or peptides. The known or novel genes, receptors, or peptidesare interesting targets for new drugs to mediate pain, and provide newinformation and novel targets for new methods of pain mediation,treatment, or prevention. The evaluation in these methods can beperformed using any number of appropriate techniques and protocols knownin the art and readily available for measuring, detecting, andidentifying genes, receptors, or peptides.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although suitable methods andmaterials are described below, methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a bar graph of force in grams (high force correlates with highpain threshold) versus time of testing, for untreated rats and ratstreated with SAN-Gly. Error bars represent one standard deviation.

FIG. 2 is a bar graph of force in grams (high force correlates with highpain threshold) versus time of testing, for untreated rats and ratstreated with SAN-Gly at 100 mg/Kg, 200 mg/Kg, and 300 mg/Kg. Error barsrepresent one standard deviation.

DETAILED DESCRIPTION

The invention relates to methods of decreasing or preventing pain byadministering to a subject a water-soluble spicamycin derivative(Formula II) in an amount sufficient to decrease or prevent the pain.Accordingly, the compound of the present invention can be administeredvia any appropriate route, e.g. intravenously, intraarterially,topically, nasally, via inhalation into the lungs, intraperitoneally,intrapleurally, orally, subcutaneously, intramuscularly, sublingually,intraepidermally, vaginally, or rectally. The compound can be formulatedas a solution, suspension, suppository, tablet, granules, powder,capsules, ointment, or cream. A variety of additives can be added tothese formulations, such as a solvent (e.g., water or physiologicalsaline), solubilizing agent (e.g., ethanol, Polysorbates, or CremophorEL7®), agent for achieving isotonicity, preservative, antioxidizingagent, excipient (e.g., lactose, starch, crystalline cellulose,mannitol, maltose, calcium hydrogen phosphate, light silicic acidanhydride, or calcium carbonate), binder (e.g., starch,polyvinylpyrrolidone, hydroxypropyl cellulose, ethyl cellulose, carboxymethyl cellulose, or gum arabic), lubricant (e.g., magnesium stearate,talc, or hardened oils), or stabilizer (e.g., lactose, mannitol,maltose, polysorbates, macrogels, or polyoxyethylene hardened castoroils). If suitable, the following compounds can also be added: glycerin,dimethylacetamide, sodium lactate, a surfactant, or a basic substancesuch as sodium hydroxide, ethylenediamine, ethanolamine, sodiumbicarbonate, arginine, meglumine, or trisaminomethane. As discussedabove, organic solvents (e.g., ethanol) are not required forpharmaceutical formulations containing compounds of Formula II. However,the solubilizing agents and organic materials listed above can be usedif a hydrophobic material (e.g., a second analgesic) is included in theformulation, or if the pharmacokinetic characteristic of the formulationis to be modulated. Pharmaceutical preparations such as solutions,tablets, granules, or capsules can be formed with these components orthe like.

The dose of the compound of the present invention is determined inconsideration of the results of animal experiments and variousconditions. For example, any candidate compound for pain relief can betested in the animal models described in the example below. Morespecific doses obviously vary depending on the administration method,the condition of the subject such as age, body weight, sex, sensitivity,food eaten, dosage intervals, medicines administered in combination, andthe source, seriousness, and degree of pain. The optimal dose and theadministration frequency under a given condition must be determined bythe appropriate dosage test of a medical specialist based on theaforementioned guide.

Water-soluble, non-toxic, or less toxic derivatives of spicamycin, suchas SAN and SAN-Gly, can be prepared using methods known in the art. Forexample, general synthetic strategies are described in U.S. Pat. Nos.5,461,036 and 5,631,238. These strategies can be adapted to attach any Rgroup containing one or two carbons onto a sugar group, as shown inFormula II. A specific semi-synthetic strategy for preparing SAN andSAN-Gly is described in Kamishohara et al., J. Antibiotics 46:1439–1446,1993; Kamishohara et al., Oncology Res. 6:383–390, 1994; and U.S. Pat.Nos. 5,461,036 and 5,631,238. Synthetic chemistry transformations andprotecting group methodologies (protection and deprotection) useful insynthesizing the inhibitor compounds described herein are known in theart and include, for example, those such as described in R. Larock,Comprehensive Organic Transformations, VCH Publishers (1989); T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d.Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser andFieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); andL. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, JohnWiley and Sons (1995), and respective subsequent editions thereof.

The compounds of this invention include all salt forms thereof. Examplesof such salts include those derived from pharmaceutically acceptableinorganic and organic acids and bases. Examples of suitable acid saltsinclude acetate, adipate, alginate, aspartate, benzoate, butyrate,citrate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate,hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,lactate, maleate, malonate, methanesulfonate, nicotinate, nitrate,oxalate, palmoate, pectinate, persulfate, picrate, pivalate, propionate,salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate andundecanoate. Other acids, such as oxalic, while not in themselvespharmaceutically acceptable, may be employed in the preparation of saltsuseful as intermediates in obtaining the compounds of the invention andtheir pharmaceutically acceptable acid addition salts. Salts derivedfrom appropriate bases include alkali metal (e.g., sodium, potassium),alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)₄ ⁺ salts.Compounds of the formulae herein include those having quaternization ofany basic nitrogen-containing group therein. The compounds of thisinvention may contain one or more asymmetric centers and thus occur asracemates and racemic mixtures, single enantiomers; individualdiastereomers and diastereomeric mixtures. All such isomeric forms ofthese compounds are expressly included in the present invention.

Neuropathic pain is pain derived from a lesion or disorder of theperipheral nervous system (reviewed in Woolf, Acta Neurochir.58:125–130, 1993, and Bennett, Neuropathic Pain. In: Textbook of Pain,P. D. Wall and R. Malzack, eds., 201–224, Churchill Livingstone,Edinburgh (1994)). Patients with neuropathic pain typically present witha characteristic set of sensory disorders independent of the cause,including a constant scalding or burning pain, a partial loss ofsensitivity, tactile or cold allodynia, or hyperpathia to repeatedstimulation. Peripheral neuropathic pain includes a number of diverseconditions, the commonest of which are trigeminal neuralgia,postherpetic neuralgia, painful diabetic neurophathy, and the reflexsympathetic dystrophies including causalgia, mononeuropathies, andperipheral nerve injury.

Nociceptive pain is pain caused by an injury or disease outside thenervous system. It is often an on-going dull ache or pressure, ratherthan the sharper, trauma-like pain more characteristic of neuropathicpain. Examples of nociceptive pain include pain from cancer orarthritis, sprains, bone fractures, burns, bumps, bruises. With acutepain, the severity of pain directly correlates to the level of tissuedamage. This provides a protective reflex, such as the reflex to moveone's hand immediately if upon touching a-sharp or hot object. This typeof pain is a symptom of injured or diseased tissue, so that when theunderlying problem is cured the pain goes away. In chronic pain, thepain differs from acute pain as it does not serve a protective or otherbiological function. Rather, the nerves continue to send pain messagesto the brain even though there is no continuing tissue damage.

Due to the differences in the mechanism and types of pain, it can beadvantageous to selectively treat or prevent neuropathic painselectively over nociceptive pain. In other instances, the pain or painsymptoms can be a combination of both neuropathic and nociceptive, inthose instances treatment of both types of pain can be appropriate.

Few non-surgical alternatives exist for a patient with a disabling painresistant to opioid drugs. The methods of this invention providealternative water-soluble pain relievers to such patients. Increasedwater solubility can increase absorption into the systemic circulationwhen administered orally to a subject, thereby increasingbioavailability of a drug.

In addition, water-soluble derivatives of spicamycin, such as SAN-Gly,are less cytotoxic than water-insoluble derivatives of spicamycin, suchas KRN5500 (Kamishohara et al., Oncology Res. 6:383–390, 1994). Thisimplies that water-soluble derivatives of spicamycin are safer forpatients receiving repetitive administrations, as may be required fortreating chronic pain.

The invention also contemplates combination formulations containing awater-soluble derivative of spicamycin and a second analgesic or drug,such as an anti-inflammatory agent (e.g., aspirin, acetaminophen,ibuprofen, naproxen, diclofenac, celecoxib, NSAIDS, COX-1 inhibitors,COX-2 inhibitors, steroids, steroid derivatives, glucocorticoids).

Without further elaboration, it is believed that one skilled in the artcan, based on the above disclosure and the description below, utilizethe present invention to its fullest extent. The following example is tobe construed as merely illustrative of how one skilled in the art canpractice the invention, and is not limitative of the remainder of thedisclosure in any way. All patents and publications cited in thisdisclosure are hereby incorporated by reference.

EXAMPLE 1

To determine whether relatively water-soluble, less toxic spicamycinderivatives are useful for pain relief, the compound SAN-Gly wassemi-synthetically prepared as described in U.S. Pat. No. 5,631,238.SAN-Gly was then formulated in saline.

Male Sprague-Dawley rats (Charles River Laboratories) weighing 150–200 gwere used to evaluate the analgesic properties of the SAN-Glyformulation. The animals were housed in groups of three in plastic cageswith soft bedding and under a 12 hour light/dark cycle. Food and waterwere available ad libitum. After one week of acclimatization to thelaboratory conditions, all animals were tested to establish a baselinemechanical allodynia.

Since different animal models of pain can provide different experimentalresults, SAN-Gly was tested using two rat models. All experiments wereperformed in a single-blinded fashion, i.e., the experimenter was notaware of which rats received the control saline and which rats receivedthe SAN-Gly formulation.

One rat model used was based on an experimentally produced segmentalspinal nerve injury (Kim et al., Pain 50:355–363, 1992). One week afterthe acclimatization to laboratory conditions, baseline measurements wererecorded and surgery was performed as described in Kim et al., supra.Rats were anesthetized with halothane in oxygen and placed in a proneposition. A midline skin incision at L4–S2 was made, and paraspinalmuscles were separated from the spinous processes in the L4–S2 region.The left L5 and L6 spinal nerves were identified and tightly ligatedwith 6-O silk thread. The wound was then closed. At the end of surgery,anesthesia was discontinued, and the animals were returned to theircages with food pellets and water ad libitum. The animals recovered fromanesthesia within approximately 10 minutes.

The rats were allowed to recover from surgery for at least a week. Tento twelve days after surgery, animals were tested for allodynia bymonitoring hypersensitivity to pinprick with von Frey filaments (VFF).The animals were placed on a mesh floor and covered by a transparentplastic box open at the bottom. Calibrated VFF (3.61, 3.84, 4.08, 4.31,4.56, 4.74, 4.93, and 5.16) were applied to the plantar skin of the lefthindpaw using the up-down method and a 50% foot withdrawal (pawflinching) as the pain threshold. Each VFF was tested by inserting itfrom below the rat and through the mesh floor, and applying it to thesecond, third, and fourth digits of the foot until the filament justbent (Chaplan et al., J. Neurosci. Methods 53:55–63, 1994). A trialconsisted of four repetitive VFF applications (at a frequency of one per10–15 seconds). Rats were then separated into two groups, one receivingsaline and the other receiving the SAN-Gly formulation (100 mg/kg bodyweight; single bolus in the tail vein). Both groups were then monitoredfor allodynia at various time points after surgery.

The other rat model was based on an experimentally induced chronicconstriction nerve injury (Bennett et al., Pain 33:87–107, 1988).Peripheral mononeurophathy was produced in rats as described in Bennettet al., supra. Surgery was conducted on rats under halothane anesthesiato expose the common sciatic nerve. Then loose constrictive ligatureswere placed around the nerve, the wound closed, and the animals allowedto recover for at least a week. Animals were then tested for allodynia,separated into two groups, and retested as described above.

The results using both animal models were combined and summarized inFIG. 1. The single injection of SAN-Gly resulted in a 60% recoverytowards baseline pain behavior, and this increased pain tolerancepersisted for at least 7 days after surgery. Thus, SAN-Gly proved to beuseful as an analgesic in two animal models of pain.

EXAMPLE 2

Electrophysiological recordings were done on 650 μm thick transverseslices of lumbar spinal cord (from spared-nerve injury animals) withattached L4 dorsal root that were perfused with ice cold Krebs' solution([in mM]: NaCl 117, KCl 3.6, CaCl₂ 2.5, MgCl₂ 1.2, NaH₂PO₄ 1.2, NaHCO₃25 and glucose 11) saturated with 95% O₂ and 5% CO₂. To record primaryafferent-evoked excitatory postsynaptic currents (EPSCs) from lamina II,whole cells were patch-clamped and then voltage-clamped at −70 mV.Orthodromicstimulation of L4 dorsal root was performed with a suctionelectrode attached to a constant current stimulator and graded intensitystimulation sufficient to recruit different afferent populations (Aβ,Aδ, and C-fibers) was used. Resistance of patch pipettes were 5–10 MWwhen filled with (in mM): Cs₂SO₄ 110, CaCl₂ 0.5, MgCl₂ 2, EGTA 5, HEPES5, TEA 5, ATP-Mg salt 5. Currents were amplified (Axopatch 200A),filtered at 2 kHz, digitized at 5 kHz and analyzed using pCLAMP 6 (AxonInstruments). SAN-Gly was dissolved in ice cold KREBs and bath-appliedat two concentrations (10 and 100 mM). It was observed in three out offive cells that 100 mM SAN-Gly increased the latency and reduced theamplitude of EPSCs, consistent with the hypothesis that it might play arole in inhibiting synaptic transmission in the dorsal horn. This effectwas reversed upon washout of the drug. In addition, as expected, thesame concentration of SAN had no effect. This example demonstrates thatSAN-Gly and its derivatives are useful in treating neuropathic pain.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thisinvention.

1. A method of providing pain relief, the method comprising identifyinga subject in need of pain relief; and administering to the subject anamount of a compound of Formula II, or a salt thereof, effective toprovide significant pain relief in the subject,

wherein R₁ and R₂ are different from each other and represent —H or —OH,and R represents (1) an unsubstituted alkyl having one or two carbonatoms, or (2) —H.
 2. The method of claim 1, wherein R has two carbonatoms.
 3. A method of providing pain relief, the method comprisingidentifying a subject in need of pain relief; and administering to thesubject an amount of a compound of Formula II, or a salt thereof,effective to provide significant pain relief in the subject,

wherein R₁ and R₂ are different from each other and represent —H or —OH,and wherein R is —COCH₂NH₂.
 4. The method of claim 3, wherein R₁ is —Hand R₂ is —OH.
 5. The method of claim 1, wherein R₁ is —H and R₂ is —OH.6. The method of claim 1, wherein the pain is neuropathic pain.
 7. Themethod of claim 6, wherein the pain is postherpetic neuralgia, phantomor amputation stump pain, diabetic neuropathy, acquired immunedeficiency syndrome neuropathy, back pain, visceral pain, or chronicpancreatitic neuropathy.
 8. The method of claim 1, wherein the pain isopioid-resistant.
 9. The method of claim 1, wherein the subject is amammal.
 10. The method of claim 1, wherein the subject is a human. 11.The method of claim 1, wherein the compound is administeredsystemically.
 12. The method of claim 1, wherein the compound isadministered at a site of pain in the subject.
 13. The method of claim1, wherein the compound is administered via an implant.
 14. The methodof claim 13, wherein the implant provides slow release of the compound.15. The method of claim 1, wherein the compound is administeredintravenously.
 16. The method of claim 1, wherein the amountadministered is about 1 ng to 4 mg/m² body surface area.
 17. The methodof claim 1, wherein the amount administered is about 80 ng to 1 mg/m²patient body surface area.
 18. The method of claim 1, wherein the amountadministered is about 10 to 100 mg/kg body weight.
 19. The method ofclaim 18, wherein the amount administered is about 100 mg/kg bodyweight.
 20. The method of claim 1, wherein the compound is administeredin an aqueous solution.
 21. The method of claim 1 wherein neuropathicpain is selectively relieved over nociceptive pain.
 22. The method ofclaim 3, wherein the pain is neuropathic pain.
 23. The method of claim22, wherein the pain is postherpetic neuralgia, phantom or amputationstump pain, diabetic neuropathy, acquired immune deficiency syndromeneuropathy, back pain, visceral pain, or chronic pancreatiticneuropathy.
 24. The method of claim 3, wherein the pain isopioid-resistant.
 25. The method of claim 3, wherein the subject is amammal.
 26. The method of claim 3, wherein the subject is a human. 27.The method of claim 3, wherein the compound is administeredsystemically.
 28. The method of claim 3, wherein the compound isadministered at a site of pain in the subject.
 29. The method of claim3, wherein the compound is administered via an implant.
 30. The methodof claim 29, wherein the implant provides slow release of the compound.31. The method of claim 3, wherein the compound is administeredintravenously.
 32. The method of claim 3, wherein the amountadministered is about 1 ng to 4 mg/m² body surface area.
 33. The methodof claim 3, wherein the amount administered is about 80 ng to 1 mg/m²patient body surface area.
 34. The method of claim 3, wherein the amountadministered is about 10 to 100 mg/kg body weight.
 35. The method ofclaim 34, wherein the amount administered is about 100 mg/kg bodyweight.
 36. The method of claim 3, wherein the compound is administeredin an aqueous solution.
 37. The method of claim 3, wherein neuropathicpain is selectively relieved over nociceptive pain.